Animal Requirements All protocols and procedures in this study were carried out in compliance with federal regulations and the guidelines of the Medical College of Wisconsin’s Institutional Animal Care and Use Committee. A total of 12 male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were used in this study.
Following an acclimation period of 1 week, 12 male Sprague-Dawley rats, weighing 250–300 g, were separated into two groups (7
C7 nerve stimulation, 5
radial nerve stimulation). Group I rats underwent fMRI during direct electrical stimulation of the right C7 nerve root. Group II rats underwent fMRI during direct electrical stimulation of the right radial nerve.
Nerve Operation All 12 animals were subjected to nerve surgery following acclimation. To induce general anesthesia, the animal was first placed in a transparent chamber, where Isoflurane (1.4%, Halocarbon Laboratories, River Edge, NJ, USA) was administered through a vaporizer. The animal was then transferred to a heated operating table where isoflurane was continuously administered through a nosecone. After being placed in the supine position, the right neck and axilla were clipped with an electric razor. The area was then prepped with povidone–iodine and draped in sterile fashion. To begin the procedure, a 2.0-cm incision was placed longitudinally in the right neck, extending obliquely into the right axilla. The subcutaneous tissues were then divided to the level of the pectoralis major. The brachial plexus was then identified at the level of the terminal branches, immediately lateral to the border of the pectoralis major muscle. For animals undergoing C7 nerve electrode placement, the pectoralis major muscle was bluntly divided parallel to its fibers. The nerve roots were then identified deep to the muscle. Following identification, the C7 nerve root was isolated, and a 125-μm-diameter stainless steel bipolar electrode (Plastics 1, Roanoke, VA, USA) was placed circumferentially around the nerve root (see Fig. ). The bipolar electrodes used are coated with polymide insulation. The polymide coating was removed only on the surface of the electrode in direct contact with the nerve being tested, thereby shielding adjacent nerves. For animals undergoing radial nerve electrode placement, the dissection was performed at the level of the terminal branches of the plexus. The radial nerve was identified deep to the median and ulnar nerves. After isolation of the nerve, an electrode was placed circumferentially around the radial nerve (see Fig. ). All nerve dissections and electrode placements were performed with the use of the operating microscope. Following electrode placement, the overlying skin was closed using interrupted 3–0 nylon sutures. The pedestal of the implantable electrode was left exposed through the skin. A cable was attached to the pedestal to allow for direct nerve stimulation. At this point, venous access was obtained using direct cut-down to the right femoral vein. Tracheostomy was then performed to allow for mechanical ventilation during scanning. Immediately following the operation, the animal was transferred to the Bruker 9.4-T small-animal MRI scanner, where fMRI was performed during direct nerve stimulation.
a Close-up photograph of implantable electrode placed on the C7 nerve root. b Close-up photograph of implantable electrode placed on the radial nerve
Following the surgical procedure, once the animal was transferred to the MRI scanner, isoflurane was discontinued and dexmedetomidine (Orion Corp., Espoo, Finland) was administered intravenously at a rate of 100 mcg/kg/h, along with pancuronium bromide (Hospira, Inc., Lake Forest, IL, USA) at rate of 2 mg/kg/h [24
]. Once scanning was complete, the animal was euthanized using Nembutal (Schering-Plough Animal Health Corp., Union, NJ, USA) 120 mg/kg given intravenously.
Functional MRI Parameters
The BOLD response to nerve stimulation in the primary sensory and motor regions was studied. A rapid acquisition with relaxation enhancement (RARE) anatomic image was obtained prior to each fMRI experiment. RARE parameters were repetition time (TR)
2.5 s, echo time (TE)
50.8 ms, field of view (FOV)
35 mm, and a 256
256 matrix. Gradient recalled echo-planar scans were acquired using a Bruker AVANCE 9.4-T MRI scanner (AVANCE, Bruker, Billerica, MA, USA) with a 30-cm bore. Images were acquired using a Bruker receiving surface coil (T9208) and a linear transmit coil (T10325). Ten 1-mm-thick contiguous slices were acquired, with slice 3 located directly over the anterior commissure (−0.64 mm from bregma). This slice profile allowed adequate coverage of the sensorimotor system. A T2
-weighted echo-planar imaging (EPI) sequence was used for the fMRI experiments. EPI parameters were TR
2 s, TE
18.76 ms, FOV
35 mm, and a 96
96 matrix (zero filled to a 128
128 matrix), with the same slice geometry as the RARE images. One hundred ten images were obtained during each fMRI experiment for a total acquisition time of 3 min 40 s.
Functional MRI Electrical Stimulation Method A square-wave electrical generator (S88, Grass Telefactor, Warrick, RI, USA) equipped with a constant current unit (Grass Telefactor, Warrick, RI, USA) was used for all nerve stimulations during fMRI scanning. A standard block design stimulation protocol of 40-s rest followed by three periods of 20 s on and 40 s off was used (total of 3 min 40 s; see Fig. ). Electrical stimulation was computer-controlled and triggered by a transistor–transistor logic pulse from the scanner. Direct nerve stimulation was carried out with a fixed current of 0.5 mA, frequency of 10 Hz, and duration of 1 ms.
Fig. 4 a Once the electrode is placed, the animal is transferred to the 9.4-T small-bore animal MRI scanner. b During scanning, a block design nerve stimulation protocol is performed as shown. c Ten contiguous coronal images of the cortex are obtained during (more ...)
Functional MRI Data Analysis All EPI acquisitions were then registered to an “ideal” RARE image using the Oxford Center for Functional Magnetic Resonance of the Brain’s Linear Image Registration Tool program. The EPI datasets were used to create BOLD activation maps. The activation maps are created by performing an F test on the time series with the block design as the only regressor (AFNI, 3dDeconvolve). Activation was determined using a p value threshold of 0.005 (using AFNI). Statistical significance for the number of activated voxels was determined using an unpaired t test with a p value of <0.05.
Physiologic Monitoring During MRI Image Acquisition During all MRI scanning sessions, the rat was allowed to breathe spontaneously through a nosecone with 30% FIO2. While scanning, chest respiration (Model 1025, SA Instruments, Stony Brook, NY, USA), end tidal gases (POET IQ2, Criticare Systems, Waukesha, WI, USA), and pulse oximetry (Model 8600 V, Nonin Medical, Plymouth, MN, USA) were continuously monitored.